Name: dissection of single skeletal muscle fibers for immunofluorescent and morphometric analyses of whole-mount neuromuscular junctions
Uploaded: 2021-08-14T14:30:00
Description: Watch this Scientific Journal Video about Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions at JoVE.com

Many thanks to CSIC and PEDECIBA for the financial support given to this work; to Natalia Rosano for her manuscript corrections; to Marcelo Casacuberta that makes the video and to Nicol&#225;s Bolatto for lending his voice for it.

All animal procedures were performed according to the guidelines of the National Law N&#176; 18611 for Care of Animals Used for Experimental Purposes. The protocol was approved by the Institutional Ethical Committee (CEUA IIBCE, Protocol Number 004/09/2015).
1. Muscle dissection (Day 1)
NOTE: Before starting, make 40 mL of 0.5% paraformaldehyde (PFA), pH 7.4 in Dulbecco&#180;s phosphate saline (DPBS). Optionally, make 20 mL of 4% PFA. Prepare 5 mL aliquots and freeze ...

In this article, we present a detailed protocol for the dissection of two rat skeletal muscles (one slow-twitch and the other fast-twitch), fiber muscle isolation and immunofluorescence detection of pre and postsynaptic markers to quantitatively assess&#160;NMJ changes as well as assembly/disassembly processes. This kind of protocol can be useful in rodent models41,42 to evaluate NMJ during physiological or pathological processes as exemplified here in a model of...

The mammal neuromuscular junction (NMJ) is a large cholinergic tripartite synapse made up of the motor neuron nerve ending, the postsynaptic membrane on the skeletal muscle fiber, and the terminal Schwann cells1,2,3. This synapse exhibits high morphological and functional plasticity4,5,6,7,8, even during adulthood when NMJs can undergo dynamic structural modifications. For example, some researchers have shown that motor nerve endings continually change their shape at the micrometer scale9. It has also been reported that the morphology of the NMJ responds to functional requirements, altered use, aging, exercise, or variations in locomotor activity4,10,11,12,13,14,15. Thus, training and lack of use represent&#160;essential stimulus to modify some characteristics of the NMJ, such as its size, length, dispersion of synaptic vesicles and receptors, as well as nerve terminal branching14,16,17,18,19,20.
Furthermore, it has been shown that any structural change or degeneration of this vital junction could result in motor neuron cell death and muscle atrophy21. It is also thought that altered communication among nerves and muscles could be responsible for the physiological age-related NMJ changes and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that constitutes one of the best examples of impaired muscle-nerve interplay3. Despite the numerous studies conducted on motor neuron dysfunction, it is still debated whether the deterioration observed in ALS occurs due to the direct damage into the motor neuron and then extends to the cortico-spinal projections22; or if it should be considered as a distal axonopathy where degeneration begins in the nerve endings and progresses toward the motor neuron somas23,24. Given the complexity of ALS pathology, it is logical to consider that a mix of independent processes occurs. As NMJ is the central player of the physiopathological interplay between muscle and nerve, its destabilization represents a pivotal point in the origin of the disease that is relevant to be analyzed.
The mammal neuromuscular system is functionally organized into discrete motor units, consisting of a motor neuron and the muscle fibers that are exclusively innervated by its nerve terminal. Each motor unit has fibers with similar or identical structural and functional properties25. Motor neuron selective recruitment allows optimizing muscle response to functional demands. Now it is clear that mammalian skeletal muscles are composed of four different fiber types.&#160;Some muscles are named according to the characteristics of their most abundant fiber type. For example, the soleus (a posterior muscle of the hind limb involved in the maintenance of the body posture) bears a majority of slow-twitch units (type 1) and is recognized as a slow muscle. Instead, extensor digitorum longus&#160;(EDL) is essentially composed of units with similar fast-twitch properties (type 2 fibers) and is known as a fast muscle specialized for phasic movements needed for locomotion. In other words, although adult muscles are plastic in nature due to the hormonal and neural influences, its fiber composition determines the capacity to perform different activities, as seen in soleus that experiences continuous low-intensity activity and EDL that exhibits a more rapid single twitch. Other features that are variable among different types of muscle fibers are related to their structure (mitochondrial content, extension of sarcoplasmic reticulum, thickness of the Z line), myosin ATPase content, and myosin heavy chain composition26,27,28,29.
For rodent NMJs, there are significant differences among muscles28,29. Morphometric analyses performed in soleus and EDL from rats revealed a positive correlation between the synaptic area and fiber diameter (i.e., the synaptic area in soleus slow fibers is greater than in EDL fast fibers) but the ratio between NMJ area and fiber size is similar in both muscles30,31. Also, in relation to the nerve terminals, the endplate absolute areas in type 1 fibers were lower than in type 2 fibers, whereas the normalization by fiber diameter made areas of nerve terminals in type 1 fibers the largest32.
However, very few studies focus on morphometric analysis to show the evidence of changes in some of the NMJ components33,34. Thus, due to the relevance of the NMJ in the function of the organism, whose morphology and physiology are altered in various pathologies, it is important to optimize dissection protocols of different types of muscles with quality enough to allow the visualization of the whole NMJ structure. It is also necessary to evaluate the occurrence of pre or postsynaptic changes in different experimental situations or conditions such as aging or exercise35,36,37,38. In addition, it can be helpful to evidence more subtle alterations in NMJ components such as altered neurofilament phosphorylation in the terminal nerve endings as reported in ALS39.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stereomicroscope with cool light illumination</td>
			<td>Nikon</td>
			<td>SMZ-10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking platform</td>
			<td>Biometra (WT 16)</td>
			<td>042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses (24 x 32 mm)</td>
			<td>Deltalab</td>
			<td>D102432</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium (Plus) microscope slides</td>
			<td>PORLAB</td>
			<td>PC-201-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>F.S.T</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Uniband LA-4C Scissors 125mm</td>
			<td>E.M.S</td>
			<td>77910-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Disponsable surgical blades #10</td>
			<td>Sakira Medical</td>
			<td>1567</td>
			<td></td>
		</tr>
		<tr>
			<td>Disponsable sterile syringe (1 ml)</td>
			<td>Sakira Medical</td>
			<td>1569</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP pen</td>
			<td>E.M.S</td>
			<td>71310</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;l or 200 &#956;l pipette</td>
			<td>Finnpipette</td>
			<td>9400130</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 800 - AiryScan</td>
			<td></td>
		</tr>
		<tr>
			<td>NTac:SD-TgN(SOD1G93A)L26H rats</td>
			<td>Taconic</td>
			<td>2148-M</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS (Dulbecco)</td>
			<td>Gibco</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>167</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Bio Basic INC.</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Mallinckrodt</td>
			<td>5092</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Amresco</td>
			<td>497</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-Neurofilament H (NF-H), Phosphorylated Antibody</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Previously Covance # SMI 31P</td>
		</tr>
		<tr>
			<td>Purified anti-Neurofilament H (NF-H), Nonphosphorylated Antibody</td>
			<td>BioLegend</td>
			<td>801701</td>
			<td>Previously Covance # SMI-32P</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-Mouse IgG (H+L)</td>
			<td>Thermo Scientific</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Bungarotoxin, biotin-XX conjugate</td>
			<td>Invitrogen</td>
			<td>B1196</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 555 conjugate</td>
			<td>Invitrogen</td>
			<td>S32355</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminophenylindole (DAPI)</td>
			<td>Sigma</td>
			<td>D8417</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of single skeletal muscle fibers for immunofluorescent and morphometric analyses of whole-mount neuromuscular junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

This protocol offers a straightforward method to isolate and immunostain muscle fibers from two different types of muscles (fast- and slow-twitch muscles, see Figure 1). Using the correct markers and / or probes, NMJ components can be detected and evaluated since a quantitative point of view to assess some of the morphological changes that can occur as consequence of illness progression or a specific drug treatment. In the present study, presynaptic and postsynaptic components of the NMJ wer...

Watch this Scientific Journal Video about Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions at JoVE.com

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Research

JoVE Journal

Neuroscience

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Single Drosophila Ommatidium Dissection and Imaging

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...